PROJECT SUMMARY The packaging of DNA into chromatin makes it largely inaccessible for the central nuclear processes of transcription, replication, recombination and repair. ATP-dependent chromatin remodeling motors play key roles in both increasing DNA access within chromatin as well in generating higher-order chromatin structures that promote transcriptional repression. However, the mechanisms by which chromatin remodeling motors function are largely unknown. The overall goal of this proposal is to use concepts learnt from well-studied motors such as kinesin and helicases to understand the how a major ATP- dependent chromatin-remodeling complex, human ACF functions. ACF generates evenly spaced nucleosomes to enable higher-order chromatin folding and gene silencing. Our work over the last grant period has shown that ACF functions as a dimeric motor in which each ATPase subunit takes turns engaging either side of a nucleosome. Here we will build on these discoveries to address the following questions: (1) How are the activities of the two ATPase subunits in ACF coordinated? (2) How does ACF recognize and use specific nucleosomal features in its reaction mechanism? (3) How is ACF activity regulated by the presence of adjacent nucleosomes?